Utilising loquat leaf extract for hair growth stimulation

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In this study, we searched for a number of plant extracts for hair growth, and found that loquat (Eriobotrya japonica) (Lindley) leaf extract and its key component, corosolic acid, are promising ingredients.

RT-PCR analysis of hair growth cycle related cytokines showed loquat leaf extract and corosolic acid up-regulated the mRNA expression levels of FGF-7, VEGF, BMP-2 and down-regulated that of FGF-5 in dermal papilla cells. It suggests that the loquat leaf extract and corosolic acid relates to hair growth by regulation of several growth factors in dermal papilla cells. We show here the molecular regulation of hair growth factors and cytokines by loquat leaf extract and corosolic acid, and describe the possibility for hair growth material.

Materials and methods

Preparation of plant extract and purification of corosolic acid
The dried leaves of E. japonica (Fig. 1) were extracted with 80% EtOH under reflux for two hours. The loquat leaf extract was obtained after an activated charcoal treatment and concentration. It was purified using ODS-column, SiO2 column and finally by HPLC to give corosolic acid as 2.5% yield from the loquat leaf extract (Fig. 2).

Cells
Human follicle dermal papilla cells (DPCs) (Toyobo, Tokyo, Japan) and normal human epidermal keratinocytes (NHEK) (Kurabo Industries, Osaka, Japan) were purchased and cultured with PCGM (Toyobo) and Epilife-KG2 (Kurabo Industries) containing growth supplements, respectively.

Reverse transcription polymerase chain reaction (RT-PCR)
DPCs were treated for two hours in FBSfree DMEM, together with the indicated concentrations of loquat leaf extract and corosolic acid. Total RNA was isolated and semi-quantitative RT-PCR was performed by Thermal Cycler (TaKaRa Bio, Otsu, Japan). The 0.5 microgram of total RNA was reverse-transcribed. The primers used for amplifying the respective fragments are listed in Table 1. PCR products were quantified using Kodak 1D Image Analysis Software after agarose gel electrophoresis.

Results

Loquat leaf extract and corosolic acid induced the proliferation of DPCs and NHEK
Loquat leaf extract and corosolic acid increased the proliferation of cultured human DPCs (Fig. 2) and NHEK (Fig. 3). Four days’ treatment with various concentrations of loquat leaf extract increased the DPCs proliferation significantly (p<0.05); 112% and 122% at 2.5 µg/mL and 10 µg/mL, respectively. Three days’ treatment with various concentrations of loquat leaf extract also increased the NHEK proliferation significantly (p<0.05); 112% and 113% at 0.156 µg/mL and 0.625 µg/mL, respectively. Corosolic acid enhanced the significant proliferation of DPCs and NHEK at 1 µg/mL and 0.0625 µg/mL, respectively.

Effects of loquat leaf extract and corosolic acid on growth factors’ gene expression
It has been recognised that several growth factors and cytokines closely relate to the hair growth cycle. In this experiment, semi-quantitative RT-PCR analysis was performed to search for growth factors that were affected by loquat leaf extract and corosolic acid. We determined the effects of loquat leaf extract and corosolic acid on mRNA expression of FGF-7, VEGF, HGF, BMP-2, IGF-1, FGF-18 (stimulating effect on hair growth) and TGF-ß1, TGF-ß2, FGF-5 (inhibitory effect on hair growth) in DPCs. When the DPCs were treated with loquat leaf extract at dosages of 10 µg/mL and 2.5 µg/mL, the mRNA expression levels of FGF-7, VEGF and BMP-2 significantly increased as shown in Figure 5. FGF-7, VEGF and BMP-2 mRNA expressions with treatment of loquat leaf extract were up-regulated by 130%, 138% and 147%, respectively, and corosolic acid also increased the same levels that of these factors at low concentrations.

In addition, we confirmed the stimulating effect on protein level of FGF-7 (data not shown). On the other hand, the mRNA expression level of FGF-5 was down-regulated by 80% and 78% with treatment of loquat leaf extract and corosolic acid, respectively. The mRNA expression levels of HGF, IGF-1, FGF-18, TGF-ß1 and TGF-ß2, however, were not affected by loquat leaf extract and corosolic acid.

Conclusion

Many people suffer from hair loss and hair thinning, despite the development of several medical treatments, and the use of plant materials. Therefore it is important to develop new promising materials that prevent hair loss and enhance hair growth potential.

In this experiment, we found that loquat leaf extract and its key component, corosolic acid, are promising ingredients for hair growth. Loquat (Eriobotrya japonica) leaf extract has been widely used as a cosmetic ingredient because various triterpens, flavonoids, sesquiterpens and tannins have been found in the leaves of this plant, and some of these compounds have been reported to be biologically active, for instance to be anti-inflammatory, antioxidant, antitumour and astringent.

In this study we demonstrated that the loquat leaf extract and corosolic acid have outstanding hair growth promoting effects in vitro cultured DPCs and NHEK. It is well known that the hair thickness depends on the size of dermal papilla, so it is expected that DPCs proliferation leads to formation of thick and strong hair. In addition, hair follicle epithelial cells are known to derive from keratinocyte, so it is expected that NHEK proliferation leads to activating the epithelial cells and hair growth.

Various growth factors and cytokines are participated in hair growth cycle as a transition signal and/or hair shaft formation signal and play important roles in hair growth control. We investigated the effect on nine kinds of growth factors and cytokines that were affected during hair growth cycle in DPCs. FGF-7, HGF, IGF-1, VEGF and BMP-2 have stimulatory effects on elongation for anagen phase through the proliferation of hair follicle epithelial cells and/or hair shaft formation. FGF-18 induces the anagen phase from telogen phase according to the recent report. Lachgar et al have reported that VEGF also plays an important role in hair growth cycle through the improvement of vascular network. On the other hand, the expressions of TGF-ß1, TGF-ß2 and FGF-5 are known to trigger the catagen transition. The expression of TGF-ß is caused by androgen, and then TGF-ß suppresses proliferation of epithelial cells and stimulates synthesis of certain caspase. FGF-5 suppresses hair growth by inhibiting anagen proceeding and inducing the transition from anagen to catagen. We confirmed that the loquat leaf extract promotes hair growth by up-regulating FGF-7, BMP-2 (elongation of anagen) and VEGF (improvement of vascular network) and by down-regulating FGF-5 (suppression the transition from anagen to catagen). Moreover, we investigated the protein levels of these growth factors, and confirmed that loquat leaf extract and corosolic acid enhanced these proteins. Based on the abilities to stimulate DPCs and NHEK proliferation and to regulate several growth factors expression, the loquat leaf extract and corosolic acid may be good candidates for the promotion of hair growth. Loquat leaf extract and its key component, corosolic acid, have potent hair growth potential and can be used for total hair care cosmetic products.

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